Chromatin interaction is important in gene regulation. The recently completed human genome sequences provide frameworks of genetic information. However, the human genome structure and information is often presented as a one-dimensional linearity, which is short to explain the complexity and coordination of a cellular system. An entirely different perspective is required for understanding how a genome actually functions as an orchestrated system in a three-dimensional nucleus in living cells. Genomic DNA (estimated to be two meters long stretched out) is condensed in chromosomes only a few microns across in a nucleus. It is known that chromosomes are unevenly organized into euchromatins and heterochromatins, which are packaged by chromatin proteins and communicated by transcription factors for transcription and replication.
These activities appear to be ordered. It has been observed that large chromosomal loops contain active genes. Furthermore, it has been suggested that distal regulatory elements such as locus control regions (LCR), enhancers, and insulators act by repositioning specific genetic loci to regions with active or silent transcription. Recent work has demonstrated in β-globin, and most recently, in cytokine gene (IFN-γ) that LCRs may directly interact with promoters in long distances on the same chromosome and even interact with promoters on different chromosomes. It is possible that intra- and inter-chromosomal interactions are general phenomena occurring at multiple genetic loci in coordinating gene regulation of important pathways. Inter-chromosomal interactions have also been implicated in diseases. For example, dysregulation of myc transcript is achieved by chromosomal translocations that juxtapose the c-myc/pvt-1 locus on chromosome 15 with one of the immunoglobulin loci on chromosome 12. Further analysis of interchromosomal interactions at whole genome level is necessary to identify all interactions, and will shed light on high-order gene regulations in cells.
Technologies used for studies of chromatin interactions—A number of approaches have been used to study the three-dimensional structure and chromatin interactions, all with considerable limitations. Technologies applicable to this question may be roughly classified as visualization tools, such as microscopy, Fluorescence In Situ hybridization (FISH), and RNA-TRAP (RNA Tagging and Recovery of Associated Proteins); and molecular methodologies, such as chromosome conformation capture (3C), and 3C followed by chromatin immunoprecipitation (3C-ChIP).
Microscopy was used in many early studies to investigate chromatin spatial organization in nuclei. However, such cytogenetic approach may only provide rough segment information of chromatins in chromosomes. FISH is a significant improvement in this direction, which localizes specific genetic loci to particular physical locations on chromosomes through fluorescence labelled DNA or RNA probes hybridizing to genomic DNA. However, the resolution was very still limited. Modified from FISH, RNA-TRAP is a method that may show distal enhancers in close physical proximity with gene promoters.
Chromosome Conformation Capture (3C) was originally designed to investigate chromosomal conformation in yeast (Dekker et al, 2002), and has been used to study interactions of genetic elements that are separated in long distance and/or in different chromosomes. In 3C, DNA-protein (chromatin) structures are formaldehyde cross-linked in vivo, and chromatins are fragmented by restriction enzyme digestion. DNA fragments tethered by DNA binding proteins are then joined together by ligation, and the junctions of two suspected known elements are detected by PCR. The detection of chromatin interactions mediated by specific DNA binding protein or transcription factors may be further enhanced by chromatin immunoprecipitation (3C-ChIP), in which the chimerical DNA fragments cross-linked with protein resulted in the 3C procedure are enriched by antibody pull-down.
Though each individual technique and some combinations have been demonstrated to be useful in identifying some specific intra- and inter-chromosomal interactions, these approaches relied on existing knowledge or conjecture as to what possible distal chromatin interactions may be present and primers designed to detect such junctions by PCR one region at a time. Therefore, the current technologies for study of chromatin interactions are extremely limited for identification of novel chromatin interactions and large scale at whole genome level.
Despite considerable interest in the way that chromosomes are spatially organized within the nucleus and how that may regulate transcription of distal genes in concert, only scattered and indirect information is currently available. The lack of information in this aspect is largely due to the lack of robust technologies that may effectively address three-dimensional questions of chromosomal interactions.
There is a need in the art for more efficient methods and robust technologies that may effectively address three-dimensional questions of chromosomal interactions that may overcome the disadvantages and limitations of the existing art.